Fine mapping of bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) neutralizing epitopes by type-specific monoclonal antibodies and sequence comparison with BHV-5 gD

Abstract

Overlapping fragments of the bovine herpesvirus-1 (BHV-1) glycoprotein (gD) ORF were expressed as trpE-gD fusionproteins in Escherichia coli to map linear neutralizing epitopes defined by BHV-1-specific MAbs. The MAbs 3402 and R54 reacted with the expressed fragments on Western blots that located the epitopes between the amino acids 52–126 and 165–216, respectively, of gD. Bovine covalescent sera with high neutralizing antibody titers against BHV-1 reacted with these bacterially expressed proteins containing both of the epitopes. Alignment of these sequences from BHV-1 with the corresponding region of the BHV-5 gD ORF sequences (reported here) identified several amino acid mismatches. Since the MAbs 3402 and R54 neutralize the BHV-1 and not BHV-5, it was presumed that these were important amino acids in defining the epitope. To further localize the neutralizing epitopes, synthetic peptides corresponding to these regions in the BHV-1 gD ORF were tested for their capacity to block monoclonal antibody neutralization of BHV-1 infectivity. The peptides encompassing amino acids 92—106 (3402 epitope) and amino acids 202–213 (R54 epitope) of the BHV-1 gD competed with BHV-1 for the binding by MAbs 3402 and R54, respectively, in a dose-dependent manner. Antisera produced in rabbits to these peptides conjugated to a carrier reacted strongly with a 30-kDa protein by Western blotting and had neutralizing antibody titers against BHV-1.